Dataset Information

Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease.

ABSTRACT:

Background

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons (MN) in the brain stem and spinal cord. Intracellular disruptions of cytosolic and mitochondrial calcium have been associated with selective MN degeneration, but the underlying mechanisms are not well understood. The present evidence supports a hypothesis that mitochondria are a target of mutant SOD1-mediated toxicity in familial amyotrophic lateral sclerosis (fALS) and intracellular alterations of cytosolic and mitochondrial calcium might aggravate the course of this neurodegenerative disease. In this study, we used a fluorescence charged cool device (CCD) imaging system to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells in an established cellular model of ALS.

Results

To gain insights into the molecular mechanisms of SOD1(G93A) associated motor neuron disease, we simultaneously monitored cytosolic and mitochondrial calcium concentrations in individual cells. Voltage - dependent cytosolic Ca2+ elevations and mitochondria - controlled calcium release mechanisms were monitored after loading cells with fluorescent dyes fura-2 and rhod-2. Interestingly, comparable voltage-dependent cytosolic Ca2+ elevations in WT (SH-SY5Y(WT)) and G93A (SH-SY5Y(G93A)) expressing cells were observed. In contrast, mitochondrial intracellular Ca2+ release responses evoked by bath application of the mitochondrial toxin FCCP were significantly smaller in G93A expressing cells, suggesting impaired calcium stores. Pharmacological experiments further supported the concept that the presence of G93A severely disrupts mitochondrial Ca2+ regulation.

Conclusion

In this study, by fluorescence measurement of cytosolic calcium and using simultaneous [Ca2+]i and [Ca2+]mito measurements, we are able to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells an established cellular model of ALS. The primary goals of this paper are (1) method development, and (2) screening for deficits in mutant cells on the single cell level. On the technological level, our method promises to serve as a valuable tool to identify mitochondrial and Ca2+-related defects during G93A-mediated MN degeneration. In addition, our experiments support a model where a specialized interplay between cytosolic calcium profiles and mitochondrial mechanisms contribute to the selective degeneration of neurons in ALS.

SUBMITTER: Jaiswal MK

PROVIDER: S-EPMC2716351 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Publications

Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease.

Jaiswal Manoj Kumar MK Zech Wolf-Dieter WD Goos Miriam M Leutbecher Christine C Ferri Alberto A Zippelius Annette A Carrì Maria Teresa MT Nau Roland R Keller Bernhard U BU

BMC neuroscience 20090622

<h4>Background</h4>Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons (MN) in the brain stem and spinal cord. Intracellular disruptions of cytosolic and mitochondrial calcium have been associated with selective MN degeneration, but the underlying mechanisms are not well understood. The present evidence supports a hypothesis that mitochondria are a target of mutant SOD1-mediated toxicity in familial amyotrophic lateral sc ...[more]

PMID: 19545440

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Project description:The NLRP3 inflammasome signaling pathway is a major contributor to the neuroinflammatory process in the central nervous system. Oxidative stress and mitochondrial dysfunction are key pathophysiological processes of many chronic neurodegenerative diseases, including Parkinson's disease (PD). However, the inter-relationship between mitochondrial defects and neuroinflammation is not well understood. In the present study, we show that impaired mitochondrial function can augment the NLRP3 inflammasome-driven proinflammatory cascade in microglia. Primary mouse microglia treated with the common inflammogen LPS increased NLRP3 and pro-IL-1? expression. Interestingly, exposure of LPS-primed microglial cells to the mitochondrial complex-I inhibitory pesticides rotenone and tebufenpyrad specifically potentiated the NLRP3 induction, ASC speck formation and pro-IL-1? processing to IL-1? in a dose-dependent manner, indicating that mitochondrial impairment heightened the NLRP3 inflammasome-mediated proinflammatory response in microglia. The neurotoxic pesticide-induced NLRP3 inflammasome activation was accompanied by bioenergetic defects and lysosomal dysfunction in microglia. Furthermore, the pesticides enhanced mitochondrial ROS generation in primary microglia, while amelioration of mitochondria-derived ROS by the mitochondria-targeted antioxidant mito-apocynin completely abolished IL-1? release, indicating mitochondrial ROS drives potentiation of the NLRP3 inflammasome in microglia. Exposure to conditioned media obtained from mitochondrial inhibitor-treated, LPS-primed microglial cells, but not unprimed cells, induced dopaminergic neurodegeneration in cultured primary mesencephalic and human dopaminergic neuronal cells (LUHMES). Notably, our in vivo results with chronic rotenone rodent models of PD further support the activation of proinflammatory NLRP3 inflammasome signaling due to mitochondrial dysfunction. Collectively, our results demonstrate that mitochondrial impairment in microglia can amplify NLRP3 inflammasome signaling, which augments the dopaminergic neurodegenerative process.

Project description:Parvalbumin (PV) is a cytosolic Ca2+-binding protein acting as a slow-onset Ca2+ buffer modulating the shape of Ca2+ transients in fast-twitch muscles and a subpopulation of neurons. PV is also expressed in non-excitable cells including distal convoluted tubule (DCT) cells of the kidney, where it might act as an intracellular Ca2+ shuttle facilitating transcellular Ca2+ resorption. In excitable cells, upregulation of mitochondria in "PV-ergic" cells in PV-/- mice appears to be a general hallmark, evidenced in fast-twitch muscles and cerebellar Purkinje cells. Using Gene Chip Arrays and qRT-PCR, we identified differentially expressed genes in the DCT of PV-/- mice. With a focus on genes implicated in mitochondrial Ca2+ transport and membrane potential, uncoupling protein 2 (Ucp2), mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu), mitochondrial calcium uniporter regulator 1 (Mcur1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase subunit ? (Atp5b) were found to be up-upregulated. At the protein level, COX1 was increased by 31 ± 7%, while ATP-synthase subunit ? was unchanged. This suggested that these mitochondria were better suited to uphold the electrochemical potential across the mitochondrial membrane, necessary for mitochondrial Ca2+ uptake. Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit ? levels remained unaffected. Suppression of PV by shRNA in PV-expressing MDCK cells led subsequently to an increase in COX1 expression. The collapsing of the mitochondrial membrane potential by the uncoupler CCCP occurred at lower concentrations in PV-expressing MDCK cells than in control cells. In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells. Deregulation of the cytoplasmic Ca2+ buffer PV in kidney cells was counterbalanced in vivo and in vitro by adjusting the relative mitochondrial volume and modifying the mitochondrial protein composition conceivably to increase their Ca2+-buffering/sequestration capacity.

Dataset Information

Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease.

Background

Results

Conclusion

Publications

Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease.

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